High Intracellular Level of Guanosine Tetraphosphate in Mycobacterium smegmatis Changes the Morphology of the Bacterium

Abstract

Almost one-third of the world population today harbors the tubercle bacillus asymptomatically. It is postulated that the morphology and staining pattern of the long-term persistors are different from those of actively growing culture. Interestingly, it has been found that the morphology and staining pattern of the starved in vitro population of mycobacteria is similar to the persistors obtained from the lung lesions. In order to delineate the biochemical characteristics of starved mycobacteria, Mycobacteria smegmatis was grown in 0.2% glucose as a sole carbon source along with an enriched culture in 2% glucose. Accumulation of the stringent factor guanosine tetraphosphate (ppGpp) with a concomitant change in morphology was observed for M. smegmatis under carbon-deprived conditions. In addition, M. smegmatis assumed a coccoid morphology when ppGpp was ectopically produced by overexpressing Escherichia coli relA, even in an enriched medium. The Mycobacterium tuberculosis relA and spoT homologue, when induced in M. smegmatis, also resulted in the overproduction of ppGpp with a change in the bacterium's growth characteristics.Mycobacteria have emerged as a major threat to humankind, for as many as one-third of the world's population (1.7 billion) harbors the tubercle bacillus asymptomatically (18). The latent bacilli can persist in a somewhat ill-defined physiological state in pulmonary and extrapulmonary lesions for years after infection (30). These bacilli are opportunistic and can reactivate themselves when the host is immunocompromised. To add to the misery, persistors require prolonged therapy, and Mycobacterium bovis BCG vaccination has little effect in blocking reactivation of the bacteria. Hence, for improved control of tuberculosis, it is imperative to develop effective drugs to cease the propagation and persistence of these latent bacilli. This will be greatly facilitated by a better understanding of the physiological state of these latent bacteria. Although several in vitro models suggest low extracellular concentrations of oxygen to be an important cause for mycobacterial dormancy (8,10,42), the effect of this state on cellular metabolism is not clear.It has been shown that the morphology and staining pattern of an in vitro culture of mycobacteria differ from those of persistors which are obtained from the lung lesion and are chromophobic to the conventional acid-fast staining (25, 26). The former is an acid-fast and long rod-shaped bacillus, as opposed to the latter, which is non-acid fast and granular. However, these persistors can be stained after oxidizing the cell surface with periodic acid. In yet another important observation, it has been shown that the morphology and staining pattern of such persistors can be obtained in vitro by starving the Mycobacterium tuberculosis, Mycobacterium kansasii, or Mycobacterium pheli cultures on agar plates without any nutrients (26). This key observation suggests that the natural persistors may be physiologically similar to bacteria in nutritionally starved cultu...

Roles of Brucella abortus SpoT in morphological differentiation and intramacrophagic replication

“…A modified method described previously was used to examine bacterial morphology (Ojha et al, 2000;Steel et al, 2004). B. abortus wild-type, the spoT mutant and the complemented strain were cultured in Brucella broth at 37 uC for 24 h to ensure full growth.…”

“…Recent studies have reported that the stringent response is associated with morphological changes and physiological adjustment. High levels of ppGpp cause a morphological change in Mycobacterium smegmatis (Ojha et al, 2000). ppGpp acts as a second messenger to co-ordinate virulence expression as an adaptive response to amino acid starvation in Legionella pneumophila (Hammer & Swanson, 1999).…”

RelA regulates virulence and intracellular survival of Francisella novicida

“…For example, in Legionella pneumophila, (p)ppGpp triggers a switch between the avirulent replicative phase and the virulent motile phase (Hammer & Swanson, 1999), while in M. tuberculosis, (p)ppGpp accumulation is important for the transition into latency (Ojha et al, 2000;Primm et al, 2000). In addition, the expression of specific virulence genes has been shown to be modified by RelA: in Vibrio cholerae, inactivation of RelA results in decreased expression of cholera toxin and the toxin-coregulated pilus (Haralalka et al, 2003), and a Salmonella enterica spoT/relA mutant has reduced expression of HilA, the central regulator of the Salmonella pathogenicity island (SPI) 1 and SPI 2 virulence genes (Pizarro-Cerda & Tedin, 2004).…”

Synthesis of an unusual polar glycopeptidolipid in glucose-limited culture of Mycobacterium smegmatis

“…Moreover, nutritionally starved bacteria have been demonstrated to bear significant similarity with the natural persistors (Nyka, 1974). In fact, an in vitro starvation model has led to the discovery of at least two genes, sigF (Chen et al, 2000) and relA\spoT (Primm et al, 2000 ;Ojha et al, 2000) which are involved in the long term persistence of Mycobacterium tuberculosis. With an objective of gaining further insight into the physiological changes that occur during nutritional starvation in mycobacteria, we have studied the change in the profile of cell surface biologically active lipid, glycopeptidolipid (GPL), of Mycobacterium smegmatis in response to carbon starvation.…”